Germ cell depletion using HSV-TK in mouse testes

doi:10.21203/rs.3.rs-4951538/v1

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Germ cell depletion using HSV-TK in mouse testes

https://doi.org/10.21203/rs.3.rs-4951538/v1

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Germ cell transplantation is useful for the study of male germ cells and the generation of genetically modified animals. For transplantation, germ cell-free hosts generated using anticancer drug treatment, irradiation exposure, or genetic mutation are required. In this study, we aimed to develop a new system for germ cell depletion, more in compliance with the “3R” principles. For this purpose, we generated knock-in mice expressing a subtype of the herpes simplex virus type 1 thymidine kinase (HSV-TK30), reported to not induce infertility, unlike the original HSV-TK gene. Ganciclovir injection resulted in nearly complete abrogation of spermatogenesis. Furthermore, transplanted spermatogonial stem cells were differentiated into sperm in the host testes, and they gave rise to offspring. Therefore, the mice developed in this study enable the efficient removal of germ cells for germ cell transplantation in a manner more compliant with the 3R principles.

Biological sciences/Developmental biology

Biological sciences/Developmental biology/Germline development

Biological sciences/Developmental biology/Germline development/Spermatogenesis

Germ cell transplantation

Spermatogonial stem cells

Host mice

Testes

HSV-TK

DDX4

Vasa

piRNA

PIWI-piRNA

HSV-TK30 knock-in mice enable germ cell depletion in a 3R-compliant fashion.

Germ cell transplantation is useful for the generation of genetically modified animals,¹ the study of gene function during gametogenesis,² propagation of livestock with useful characteristics,³ preservation of endangered species, and fertility restoration.^4–6 Successful gametogenesis after transplantation has been observed for cultured mouse spermatogonial stem cells (SSCs) and primordial germ cell-like cells.^7,8 In livestock animals, transplantation of in vivo SSCs results in spermatogenesis from these cells ⁹. For the survival of transplanted germ cells in host testes, it is essential to deplete endogenous germ cells, as they compete with transplanted germ cells for the niche.¹⁰

Three techniques have been used for the ablation of germ cells from mammalian testes. The most common method is busulfan injection, an alkylating agent that affects proliferating spermatogonia.^11–14 However, busulfan also targets other dividing cells, induces hematopoietic toxicity, and inflicts pain.¹⁵ The second method is irradiation in which damage is restricted to the scrotal region, while other regions are protected.^16–19 Nevertheless, this method is sometimes inconvenient as it requires specific instrumentation. The third method is the use of genetically modified animals that lack germ cells. The most common genetically modified host is the c-Kit mutant mouse (W/W^v trans heterozygote).¹² However, only breeding between heterozygous mice (+/W × +/W^v) results in mice with the expected genotype with a probability of 25%, because homozygotes and trans heterozygotes are lethal and infertile, respectively. Nanos2 KO mice can also be used for the host animals. This has been applied to pigs, goats, and bovines ⁹. The pair between heterozygous males and homozygous females can produce the expected males with a probability of 50%. However, many unnecessary males are produced.

The HSV-TK/ganciclovir system has been used in conditional ablation of only a predetermined cell type expressing HSV-TK.^20,21 In contrast to cellular thymidine kinase, HSV-TK reacts with a wide range of substrates including purine analogs.^22,23 Therefore, cells expressing HSV-TK are susceptible to nucleoside prodrugs, such as ganciclovir.^24,25 Ganciclovir is converted to ganciclovir monophosphate by HSV-TK. Ganciclovir monophosphate is subsequently phosphorylated to produce ganciclovir triphosphate by cellular kinase, which resembles guanosine triphosphate (GTP). During cell proliferation, it is incorporated into DNA, instead of GTP, and replication is inhibited, eventually leading to cell death.²⁶

Animal research ethics describes the internationally accepted “3R principles” (replacement, reduction, and refinement), which provide a framework for conducting humane animal experiments. Considering these principles, we embarked on a new approach that is more compatible with them. In the present study, we utilized the HSV-TK/ganciclovir system to specifically deplete germ cells. SSC transplantation into the germ cell-depleted host mice resulted in successful generation of offspring. Overall, our results show that the mice developed in this study can be used for germ cell transplantation.

Generation of knock-in mice

To deplete male germ cells using HSV-TK, we inserted the P2A self-cleaving peptide and HSV-thymidine kinase (HSV-TK) gene into the C-terminus of the germ cell-specific gene, Vasa (a.k.a. Ddx4 or Mvh) (Fig. 1a). Because male infertility is caused by the short product of HSV-TK that is generated from the testis-specific cryptic promoter in the coding region of HSV-TK,²⁷ we utilized an HSV-TK containing five-point mutations (HSV-TK30).²² Male mice harboring HSV-TK30, unlike regular HSV-TK, have been reported to generate offspring.²⁸

Vasa^wt/Tk30 and Vasa^Tk30/Tk30 female mice were fertile (Table S1). Male Vasa^wt/Tk30 mice also produced normal-sized litters (Table S1). In contrast, male Vasa^Tk30/Tk30 mice were infertile, and few, if any, sperm were observed in the epididymis of the Vasa^Tk30/Tk30 mice (Fig. 1b). Furthermore, Vasa^Tk30/Tk30 testes contained a smaller number of elongated spermatids (59.5 elongated spermatids per tubule on average) compared with Vasa^wt/wt (101.6) and Vasa^wt/Tk30 testes (103.3) (Fig. 1b, 1c). A close inspection revealed that the heads of the elongated spermatids in the Vasa^Tk30/Tk30 testes seemingly failed to be fully elongate (Fig. 1d). The difference first became clear in stage XII tubules. Consistent with the notion that only the last step of sperm morphogenesis is affected, qPCR analysis did not show any significant differences in the expression levels of different markers between the three genotypes (Fig. 1e).^29–36

As homozygous Mvh null mutation has reported to result in arrest of spermatogenesis at the spermatocyte stage,³⁷ the spermatid arrest phenotype of Vasa^Tk30/Tk30 mice suggests that the TK30 locus produces at least partially functional Mvh protein. We considered the following two possible mechanisms for the spermatogenic defects observed in Vasa^Tk30/Tk30 testes: (1) hypomorphic mutation for Mvh functions, (2) deleterious effects of the high level of TK30 expression. To distinguish these two possibilities, we introduced the Mvh-null allele to generate Vasa^Tk30/- mice. No elongated spermatids were observed in Vasa^Tk30/- mouse testes (Fig. 2a). PAS-staining analyses of acrosome formation revealed that round spermatids were arrested at step I or II (Fig. 2b). The qPCR analyses showed that Prm2 expression was decreased in Vasa^Tk30/- mouse testes (Fig. 2c). Vasa^Tk30/- mice showed an intermediate phenotype between Vasa^Tk30/Tk30 and Vasa^-/- mice. The results favor the hypomorphic mutation mechanism.

Required dosage of ganciclovir for complete germ cell depletion

To determine the dose of ganciclovir required to successfully deplete germ cells, 3-week-old Vasa^wt/Tk30 and Vasa^wt/wt mice were injected with seven different doses of ganciclovir (0.5, 2, 5, 15, 150, 400, 1000 mg/kg, single injection). After 6 weeks, the testes were harvested. In testes injected with 0.5 mg/kg of ganciclovir, no marked difference was observed between Vasa^wt/wt and Vasa^wt/Tk30 (Fig. 3a, 3b). In contrast, the injection of 2 mg/kg of ganciclovir in Vasa^wt/Tk30 mice resulted in significantly smaller and lighter (3.06-fold difference) testes compared with those of Vasa^wt/wt mice (Fig. 3a, 3b). With the 2 mg/kg dose, spermatids were found in 60.3% of the Vasa^wt/Tk30 tubules (Fig. 3c, 3d), although spermatocytes were present in mostly all (95.9%) seminiferous tubules. With the 5 mg/kg dose, spermatocytes were found only in 10% of tubules. Remarkably, when Vasa^wt/Tk30 mice were injected with 15 mg/kg of ganciclovir, more severe effects were observed (Fig. 3a-d). Spermatocytes were present only in 0.55% of the tubules. Almost none of the tubules contained spermatocytes with doses of 150, 400, and 1000 mg/kg (Fig. 3d). However, dramatic decrease in testicular weight was observed even in the control Vasa^wt/wt testes when high doses of ganciclovir (≥ 400 mg/kg) were injected (Fig. 3b). Spermatocytes were observed only in 14.5% of Vasa^wt/wt tubules with the 400 mg/kg dose (Fig. 3d, 3e). When 1000 mg/kg was injected into Vasa^wt/wt mice, all tubules lacked spermatocytes and spermatids. A very high dose of ganciclovir injection is likely harmful to animals.

To determine whether ganciclovir injection similarly affected mature adult mice, 2, 15, and 150 mg/kg ganciclovir was injected into Vasa^wt/wt and Vasa^wt/Tk30 mice. The testes were then harvested after 6 weeks. With 2 and 15 mg/kg doses, Vasa^wt/Tk30 mice exhibited smaller testes compared with the Vasa^wt/wt mice (Fig. 4a, 4b). However, with the 150 mg/kg dose, not only Vasa^wt/Tk30 but also Vasa^wt/wt testes were similarly shrunken. Histological analysis revealed that spermatocytes and spermatids were absent in 90.1% (2 mg/kg), 100% (15 mg/kg), and 98.7 (150 mg/kg) of seminiferous tubules of Vasa^wt/Tk30 testes (Fig. 4c). When 150 mg/kg of ganciclovir was injected, most (92.2%) tubules did not contain spermatocytes and spermatids even in Vasa^wt/wt mice.

To examine the long-term recovery of spermatogenesis after the ganciclovir injection, testes were harvested 3 months after injection (15 or 150 mg/kg ganciclovir) into adult testes. In Vasa^wt/wt mice injected with 150 mg/kg, tubules showing spermatogenesis seemingly more frequently observed in 3 months compared with 6 weeks (Fig. 4b, 4c). Remarkably, almost all tubules of Vasa^wt/Tk30 mice still lacked spermatocytes and spermatids even with the 15 mg/kg dose. The effect of germ cell depletion is long-lasting in Vasa^wt/Tk30 mice.

Time course of germ cell loss

To investigate the course of germ cell depletion, adult Vasa^wt/Tk30 mice were injected with 15 mg/kg of the drug and the testes were harvested at Day 4, Day 14, and Day 25. On Day 4, leptotene spermatocytes were largely depleted in the stage VIII tubules (Fig. 5a, black arrows in the control section show leptotene spermatocytes). Consistent with this, qPCR analyses revealed that Stra8 expression was dramatically reduced (Fig. 5b). In Day 14 Vasa^wt/Tk30 testes, few or no spermatocytes were observed, whereas all seminiferous tubules contained either round spermatids or elongated spermatids (Fig. 5a). In qPCR analyses, most germ cells genes (Nanos2, Sohlh2, Stra8, Dmc1, PIWIL1) were downregulated, but a spermatid marker Prm2 was unchanged (Fig. 5b). On Day 25, about half of the seminiferous tubules contained elongated spermatids, but other germ cell types were not observed. Decrease in the expression level of Prm2 was observed (Fig. 5b). Sertoli cells were observed in all tubules at all time points (Fig. 5a, b).

Vasa^wt/Tk30 mice serve as host mice for germ cell transplantation

To examine whether Vasa^wt/Tk30 mice can be used for germ cell transplantation, cultured SSCs (GS cells) were transplanted into eight testes of Vasa^wt/Tk30 mice 10–26 days after ganciclovir (15 or 150 mg/kg) injection (Table S2). As the injected GS cells harbor CAG-EGFP transgene (hemizygote),⁸ successful transplantation can be assessed by EGFP fluorescence. Four months after the injection, four testes were harvested. Three testes showed EGFP signals in large portions (60% or more) (Table S2, Figure S1a). In these three testes, many of the tubules contained spermatocytes/spermatids (Table S2, Figure S1b). These spermatocytes/spermatids were all positive for EGFP fluorescence. In addition, some sperm were observed. The remaining four testes were harvested 9 months after the injection. EGFP was observed in the entire regions (90% or more) in three of the four testes (Fig. 6a, Table S2). In most tubules, normal spermatogenesis was observed (Fig. 6b, Table S2). All germ cells we observed in the 9-month testes were still positive for EGFP.

To determine if sperm derived from the transplanted cells can produce offsprings, testicular sperm were collected from one of the testes harvested 4 months after the injection. Intracytoplasmic sperm injection (ICSI) was performed. On the following day, ~ 41% (9/22) of the injected oocytes developed into 2-cell stage embryos. They were transferred into recipient mice. Two pups were born and one of them showed EGFP fluorescence (Figure S1c). Similarly, epididymal and testicular sperm were collected from the 9-month samples to perform intracytoplasmic sperm injection (ICSI). Approximately 47% (18/38, epididymal sperm) and 42% (8/19, testicular sperm) of oocytes developed into 2-cell stage embryos on the following day of ICSI. These 2-cell stage and 1-cell stage embryos (epididymal, 5; testicular, 1) were transplanted into the recipient oviducts. A total of six pups were obtained (epididymal, 3; testicular, 2; unknown, 1) and four of them were positive for EGFP (epididymal, 2; testicular, 1; unknown, 1) (Fig. 6c). As the transplanted GS cells were hemizygous for CAG-EGFP transgene, most, if not all, were likely derived from the transplanted cells.

Germ cell transplantation is useful for the study of spermatogenesis, generation of genetically modified animals, and the potential preservation of endangered species. In the present study, we generated host mice for germ cell transplantation by expressing HSV-TK30 in germ cells. Compared with conventional methods, our system complies with the internationally accepted “3R principles”. A single injection of 15 mg/kg of ganciclovir completely depleted testis germ cells and little or no recovery of spermatogenesis was observed after 3 months. Furthermore, transplanted SSCs were differentiated into sperm in the host testes. Using these sperm, offspring were successfully generated. Therefore, the mice developed in this study are useful for germ cell transplantation.

Our results indicated that the injection of ganciclovir at 15 mg/kg in adult mice completely depleted germ cells after 3 months without apparent harmful effects. In contrast, testes treated with busulfan exhibit some recovery after three months.³⁸ In addition, busulfan treatment is accompanied by significant toxicity risks. Although recent injection techniques, through either intratesticular injection³⁹ or two intraperitoneal injections at 3-hour intervals,³⁸ showed a significant reduction of toxicity, they require anesthesia or two treatments. Additionally, in these methods, recovery of spermatogenesis was observed after 2 or 3 months after the injection. Thus, the host system developed in the present study is more compliant with the “refinement” component.

Although it is beyond the scope of our study, another possible application of the knock-in mice developed in the present study is providing host embryos for embryonic stem (ES) cell injection. A low contribution of ES cells to the germline is a major issue when preparing ES cell chimera. Specific depletion of host germ cells enables ES cell-derived germ cells, if present, to propagate in the host testes. However, an important caveat of this approach is the bystander effect from the host germ cells. One potential method to minimize the bystander effect is to inject a minimum amount of ganciclovir such that endogenous germ cells are preferentially removed.

Vasa^Tk30/Tk30 homozygote mice showed an abnormality of spermatid elongation. The observed infertility likely results from the hypomorphic mutation of the Vasa (Mvh) gene due to addition of P2A self-cleavage peptide. Interesting, the C-terminal of Vasa is highly conserved among diverse animals from sea urchins to humans.⁴⁰ Mutation of this region in Drosophila results in severe defects in piRNA-mediated retrotransposon silencing. In mice, mutations in piRNA pathway genes, including Vasa, usually show arrest at the zygotene stage or the early round spermatid stage in spermatogenesis.^37,41 As Vasa^Tk30/Tk30 mice show spermatogenic defects at the elongating spermatid stage, Vasa^Tk30/Tk30 mice may be useful for the study of the Mvh function in the later stage of spermatogenesis.

Although this was not tested in this study, Vasa^Tk30/Tk30 homozygote mice can be similarly used as host mice for germ cell transplantation. In fact, as they are defective in producing functional sperm, offspring generated from the host mice should be derived from the transplanted cells. In summary, the mouse line developed in this study is useful for germ cell transplantation.

Animals

Animal experiments were approved by the Animal Care and Use Committee in NCCHD (A2022-008). All methods were carried out following institutional regulations. All methods are reported in accordance with ARRIVE guidelines

By using the CRISPR-Cas system, we obtained a total of 31 F0 mice (BDF1 x BDF1) and three of them had the expected genetic alteration. For the experiments, we used offspring derived from one of the three mice. One line of Vasa-mutTK knock-in founder female mice was selected for breeding with a C57BL6/J male mouse. Two F1 mice with an expected knock-in allele were then selected for breeding with C57BL6/J mice. The F2 knock-in male and female mice were crossed and their offspring were used for the experiments. Mice are available from RIKEN BRC (RBRC12307, B6;Cg-Ddx4 < em1(HSV-TK30)Twata>). Mvh mutant mice were obtained from Satomi Kuramochi-Miyagawa.

Drug injection and sample collection

To examine the effect of different concentrations of ganciclovir (Denosine, Mitsubishi Tanabe) on knock-in mice, Vasa^wt/Tk30 and Vasa^wt/wt male mice were injected with different doses of ganciclovir. The injected mice were sacrificed by cervical dislocation to collect the testes. Testes were weighed and fixed in Bouin’s solution or 4% PFA solution, washed in PBS, and placed in 70% ethanol before paraffin embedding.

Transplantation

GS cells derived from Green mice⁸ (gift from Takashi Shinohara) were cultured in IMDM/FBS medium.⁴² GS cell transplantation was performed 10 to 26 days after the injection of 15 or 150 mg/kg of ganciclovir. Approximately 1/10 volume of 0.4% Trypan blue solution (T10282, Invitrogen) was added to GS cell suspension (2.5 × 10⁷ cells/mL in IMDM). The cell suspension was kept on ice until injection. Injection needles were prepared by pulling glass capillary (G1.2 from Narishige or TW120F-4 from World Precision Instruments) using a P1000 micropipette puller (Sutter) with the following conditions: Heat = 720, Pull = 0, Vel = 85, Time = 150, Pressure = 200. Rete testes was located by inserting the needle along the efferent duct. Approximately 10–20 µL of cell suspension was injected into Rete testes using Femtojet4i (Eppendorf) using the following conditions: pi = 1000 hPa, pc = 0 hPa. Only testes in which > 60% of regions were filled with injected solution were used for analyses.

ICSI

For ICSI using sperm from testes 8 months after GS cell transplantation (see supplementary information for the 4 month samples), oocytes were collected from 10-week-old C3H/HeYoKSlc (SLC) mice by superovulation.⁴³ Oocytes before ICSI were cultured in mHTF at 37°C in 5% CO₂. For the collection of testicular sperm, a portion of testis was placed in 0.9% NaCl solution. Several incisions were made with scissors. After pipetting several times using a yellow tip with the trimmed end, 50 µL of the suspended solution was gently layered on 600 µL of 45% Percoll/0.9% NaCl solution. After centrifugation at 1677 × g at room temperature for 5 minutes, the supernatant was discarded without disturbing the approximately 30 µL of solution at the bottom, where sperm were enriched. For epididymal sperm collection, a 50 µL mHTF droplet, covered with paraffin liquid, was prepared. Cauda epididymis was put in the oil, and then several incisions were made. Using a needle, sperm were released into the mHTF drop. To prepare a microinjection chamber for ICSI, two 20 µL M2 droplets and two 20 µL 10% PVP droplets were covered with paraffin oil in a glass-bottom dish (D911600; Matsunami). Sperm were suspended in a 10% PVP droplet. ICSI was performed using a micropipette (PIN20-20FT; PrimeTech) and the Piezo-electric actuator (PrimeTech) under an inverted microscope (Ti-2U; Nikon) based on Ogonuki et al.⁴⁴ Injected oocytes were cultured for 3 hours in mHTF, then transferred to KSOM and incubated at 37°C with 5% CO₂.

Competing interests

The authors declare no competing interests.

Author Contribution

T.W. conceived the study. C.D. and T.W. designed the experiments. T.W. generated mice and performed transplantation. C.D., and S.T. performed analyses. M.S. and E.T. performed ICSI. C.D. and T.W. wrote the paper.

Acknowledgement

We thank Yusuke Shiromoto and Takashi Shinohara for GS cells, Takehiko Ogawa and Takuya Sato for SSC transplantation techniques; Hiroshi Suemizu for information regarding HSV-TK; Satomi Kuramochi-Miyagawa for MVH KO mice; Akiyumi Tashiro for technichal assistance and critical reading of the manuscript. This work was supported by KAKENHI (20H05764, 20H03177, and 22K18356), AMED (JP19gm6310010, JP20gm6310010, JP21gm6310010, and JP22gm6310010), and JST (JPMJPR228B).

Data Availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

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No competing interests reported.

Supplementary240411.docx
FIG7.png
Figure S1. Vasa^wt/Tk30recipient testes 4 months after GS cell transplantation a. Macroscopic observation of testes 4 months after GS cell transplantation. The transplanted GS cells harbored CAG-EGFP transgene. Bright field image (left) and EGFP fluorescent image (right) are shown. b. Immunohistochemical analysis of testis transplanted with GS cells. Testis shown in (a) was analyzed. c. Offspring produced by ICSI using sperm derived from testis shown in (a).

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You are reading this latest preprint version

Germ cell depletion using HSV-TK in mouse testes

Status:

Version 1

Abstract

Figures

Summary sentence

Introduction

Results

Generation of knock-in mice

Required dosage of ganciclovir for complete germ cell depletion

Time course of germ cell loss

Vasa^wt/Tk30 mice serve as host mice for germ cell transplantation

Discussion

Materials and methods

Animals

Drug injection and sample collection

Transplantation

ICSI

Declarations

Competing interests

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Germ cell depletion using HSV-TK in mouse testes

Status:

Version 1

Abstract

Figures

Summary sentence

Introduction

Results

Generation of knock-in mice

Required dosage of ganciclovir for complete germ cell depletion

Time course of germ cell loss

Vasawt/Tk30 mice serve as host mice for germ cell transplantation

Discussion

Materials and methods

Animals

Drug injection and sample collection

Transplantation

ICSI

Declarations

Competing interests

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1

Vasa^wt/Tk30 mice serve as host mice for germ cell transplantation